Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication |
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| Authors: | Fabio Lapenta Alejandro Montón Silva Renato Brandimarti Massimiliano Lanzi Fabio Lino Gratani Perceval Vellosillo Gonzalez Sofia Perticarari Alejandro Hochkoeppler |
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| Affiliation: | 1Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy;2Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy;3CSGI, University of Firenze, Via della Lastruccia 3, 50019, Sesto Fiorentino, FI, Italy;Institute of Molecular Genetics IMG-CNR, ITALY |
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| Abstract: | DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. |
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